PROJECT SUMMARY Pneumococcal pneumonia remains the leading cause of bacterial pneumonia in both children under 5 years of age and adults over 65 years of age. The standard, preventative therapy is the conjugate vaccine, Prevnar-13, which consists of an immunogenic carrier protein covalently attached to one of thirteen pneumococcal capsular polysaccharides. Although Prevnar-13 has significantly reduced the burden of pneumococcal disease, it only protects against 13 of the 90 plus pneumococcal serotypes; furthermore, current methods employed to expand the serotype coverage are notoriously slow requiring complex synthetic chemistries to link a new pneumococcal capsular polysaccharide to the immunogenic carrier protein. Over the last decade, we have been pioneering an innovative approach to conjugate vaccine development that drastically simplifies the production of glycoconjugates. This glycoengineering strategy, consisting of the exploitation of bacterial glycosylation machineries to generate ?bioconjugates?, eliminates the need of intricate chemical conjugation methods by employing conjugating enzymes to attach polysaccharides to acceptor proteins in Escherichia coli. Two conjugating enzymes, PglB and PglL, have been commercially utilized to generate bioconjugates as they are able to transfer a wide variety of polysaccharides to proteins; however, neither are able to transfer polysaccharides containing glucose at the reducing end (the first sugar of a growing polysaccharide chain). This seemingly simple observation has enormous implications as approximately 80% of pneumococcal capsules contain glucose at the reducing end. Recently, we have identified and patented the first conjugating enzyme that is able to efficiently transfer pneumococcal capsular polysaccharides containing glucose at the reducing end to an acceptor protein. Based on this observation, we will couple our novel conjugating enzyme technology with carrier proteins previously utilized in conjugate vaccine formulations, streamlining the generation of a superior pneumococcal vaccine with broader serotype coverage. Importantly, our glycoengineering strategy does not require pathogenic organisms as a source of polysaccharide nor chemical reactions to link polysaccharides to proteins. The proposed research in this phase I application will focus on (Aim 1) glycoengineering three commercial carrier proteins (exotoxin A, tetanus toxin fragment C, and CRM197) to contain a modular glycotag with pneumococcal capsular polysaccharides generating a new bioconjugate vaccine for pneumococcal serotypes 8, 9V, 14, and 15b. Subsequently (Aim 2) we will demonstrate the immunogenicity and efficacy of our pneumococcal specific bioconjugate vaccine compared to the standard preventative therapy Prevnar-13. Our next step for phase II funding is to expand the serotype coverage included in our bioconjugate vaccine, develop a large-scale purification scheme for obtaining our bioconjugate vaccine, as well as pre-clinical studies to further demonstrate the safety, potency, and efficacy of our next generation glycoengineered pneumococcal bioconjugate vaccine.